Online_Appendix - A Note on a Reformulation of the KHB Method

<p>Online_Appendix for A Note on a Reformulation of the KHB Method by Richard Breen, Kristian Bernt Karlson, and Anders Holm in Sociological Methods & Research</p

TG2 peptide substrates identified by nano-LC MS/MS.

<p>Glutamine residues targeted by TG2 are given in bold.</p><p>DA, deamidation</p><p>The 9mer core region of T-cell epitopes are underlined.</p><p>“x” indicates I or L.</p>a<p>number of protein entries in database.</p>b<p>not possible to determine which Q residue is transamidated (shown in bold and italic).</p>c<p>not possible to determine which Q residue is deamidated and which is transamidated.</p>d<p>not possible to determine whether Q2 or Q4 is targeted; however, this sequence has previously been shown to be targeted at Q4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014056#pone.0014056-Vader2" target="_blank">[17]</a>. The same Q residue is expected to be targeted in peptides #12–17.</p>e<p>identified in a sample incubated with TG2 for one minute.</p>f<p>two possible 9mer binding registers to HLA-DQ2.5.</p

Identified T-cell epitopes in wheat gluten and their observed T-cell response in celiac disease patients.

<p>Glutamine residues expected to be targeted by TG2 are given in bold.</p>a<p>Peptide PQPQLPYPQPQLPY harboring both epitopes, DQ2-α-II and DQ2-α-III, was tested.</p>b<p>Peptide LQPQQPFPQQPQQPYPQQPQ harboring both epitopes, DQ2-γ-III/DQ8-γ-I and DQ2-γ-VI, was tested.</p>c<p>Note that this peptide is not identical to peptide QQPPFSQQQQQPLPQ which was previously known as the “Glt-17” epitope. Peptide QQPPFSQQQQQPLPQ has previously been tested for T-cell response in CD patients: 3/20 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014056#pone.0014056-Vader2" target="_blank">[17]</a>.</p><p>♦Identified TG2 peptide substrates that harbor incomplete 9mer core binding regions.</p

Schematic view of the established method to enrich and analyze TG2 peptide substrates.

<p>A PTCEC digest of wheat gluten was mixed with a small amount of 5-BP which served as a substrate for TG2 in a transamidation reaction. The transamidated, biotinylated peptides were enriched from the digest using magnetic streptavidin beads, eluted with an excess of biotin and analyzed by LC-MS/MS. Database searching was performed using a database made up of all entries of <i>Triticum aestivum</i> present in the Uniprot database. In addition, MS/MS spectra were manually inspected.</p

Specific enrichment of transamidated peptides.

<p>A PTCEC digest of whole gluten was spiked with the transamidated DQ2-α-I and DQ2-γ-II peptides. MALDI-TOF spectra before (upper panel) and after enrichment (lower panel) are shown. The inset shows the four signals obtained for each of the two enriched peptides that correspond to the transamidated peptide, its sodium adduct, its potassium adduct and the adduct with two sodium ions, respectively. All signals observed in the lower mass range (up to m/z 900) were derived from matrix clusters.</p

Study protocol for analyses on the prospective association of work-related violence, and decision latitude with risk of depression in The Danish Work Life Course Cohort (DAWCO)

This protocol describes planned analyses on the longitudinal associations of work-related violence and decision latitiude with risk of clinical depression. The protocol gives details of the planned analyses to avoid post hoc decision making in the analysis of the data